Pan Youwen, Kaatz Laura
Technology Resources/Sterility Assurance, Baxter Healthcare Corporation, Round Lake, Illinois, USA.
Curr Protoc Microbiol. 2012 Nov;Chapter 2:Unit 2C.5.. doi: 10.1002/9780471729259.mc02c05s27.
This protocol was developed to utilize imaging flow cytometry (IFCM) in combination with fluorescent dyes to both enumerate and analyze morphological features of live and dead cells in a mixed live/dead bacterial sample. The fluorescent dyes used in this protocol include 5(6)-carboxyfluorescein diacetate (CFDA), which indicates the functional activity of esterase inside viable bacterial cells, and DRAQ7, a dye that exploits membrane-compromised bacterial cells to enter and stain the cell. The live cell population stained with CFDA emits a fluorescent green color while the dead cell population stained with DRAQ7 emits a fluorescent red color, which allows the two populations to be distinctively separated by the IFCM system. Additionally, the cytometer captures a clear image of each object, which can then be analyzed for morphology features. The IFCM system is able to reliably, accurately, and precisely determine a bacterial cell concentration as long as the concentration of cells in a sample is no less than 1 × 10(3) cells/ml. The two dyes, CFDA and DRAQ7, have been demonstrated to be an effective stain combination for bacterial viability analysis.
本方案旨在利用成像流式细胞术（IFCM）结合荧光染料，对混合的活/死细菌样本中的活细胞和死细胞进行计数并分析其形态特征。本方案中使用的荧光染料包括5(6)-羧基荧光素二乙酸酯（CFDA），它可指示活细菌细胞内酯酶的功能活性，以及DRAQ7，一种利用细胞膜受损的细菌细胞进入并染色细胞的染料。用CFDA染色的活细胞群体发出绿色荧光，而用DRAQ7染色的死细胞群体发出红色荧光，这使得这两个群体能够通过IFCM系统明显区分开来。此外，细胞仪会捕获每个物体的清晰图像，然后可以对其形态特征进行分析。只要样本中细胞浓度不低于1×10(3)个细胞/毫升，IFCM系统就能可靠、准确且精确地测定细菌细胞浓度。已证明CFDA和DRAQ7这两种染料是用于细菌活力分析的有效染色组合。
